title: A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci.
abstract: The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein-ubiquitin interactions.
authors.affiliation: Department of Pharmacology; University of Michigan Medical School; Ann Arbor, MI USA.
authors.forename: Corey M
authors.initials: CM
authors.lastname: Helchowski
authors.affiliation: Department of Pharmacology; University of Michigan Medical School; Ann Arbor, MI USA.
authors.forename: Laura F
authors.initials: LF
authors.lastname: Skow
authors.affiliation: Department of Pharmacology; University of Michigan Medical School; Ann Arbor, MI USA.
authors.forename: Katelyn H
authors.initials: KH
authors.lastname: Roberts
authors.affiliation: Department of Pharmacology; University of Michigan Medical School; Ann Arbor, MI USA.
authors.forename: Colleen L
authors.initials: CL
authors.lastname: Chute
authors.affiliation: Department of Pharmacology; University of Michigan Medical School; Ann Arbor, MI USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Canman
mesh_terms: D019313:BRCA1 Protein / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D056488:Histone Chaperones; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011836:Radiation Tolerance; D059767:Recombinational DNA Repair; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D019313:BRCA1 Protein; D002352:Carrier Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C485954:RAD18 protein, human; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C466234:UIMC1 protein, human; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases
keywords: ATM; BRCA1; DNA damage response; RAD18; RAP80; homologous recombination; ubiquitin